This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We propose small angle x-ray scattering experiments (SAXS) at SSRL to study the influence of H2O-D2O solvent substitution effect on the structural stability of globular proteins. Proteins naturally exist in aqueous solutions. However, it is common to dissolve proteins in heavy water for biophysical studies using various experimental techniques, e.g., infrared spectroscopy, Raman scattering, nuclear magnetic resonance, and neutron scattering, in order to avoid the difficulties involved in the measurement of proteins with the presence of H2O. To be valid, these studies assume that deuterium isotopic substitution does not influence biomolecular structure or dynamics significantly, based on the idea that D2O is the solvent that causes the mildest possible perturbation. However, experimental data, especially thermodynamic studies, have shown that solvent substitution can cause marked effects on the stability of proteins in solution. In particular, the substitution of D2O for H2O stabilizes the proteins against thermal denaturation and urea-induced denaturation. More significantly, it has been found that some proteins appear to adopt different structural conformations in D2O and H2O, with other solution conditions being identical. The small angle scattering experiment is the only technique that directly provides essential information on the global structural conformation of protein molecules in solution. It will greatly complement the thermodynamic results and contribute significantly to our understanding of deuterium isotopic effects on protein denaturation. For this proposed project, we will monitor the structural change of two model proteins, hen?s egg lysozyme and bovine serum albumin (BSA) dissolved in H2O and D2O, respectively, in the thermal denaturation and urea-induced denaturation processes, and study the influence on the denaturation behavior by the substitution of H2O with D2O.